Programmed cell death, or apoptosis, is an essential feature of growth and development, as the control of cell number is a balance between cell proliferation and cell death. Apoptosis is an active rather than a passive process, resulting in cell suicide as a result of any of a number of external or internal signals. Apoptotic cell death is characterized by nuclear condensation, endonucleolytic degradation of DNA at nucleosomal intervals ("laddering") and plasma membrane blebbing. Programmed cell death plays an essential role in, for example, immune system development and nervous system development. In the former, T cells displaying autoreactive antigen receptors are removed by apoptosis. In the latter, a significant reshaping of neural structures occurs, partly through apoptosis.
An increasing number of genes and gene products have been implicated in apoptosis. One of these is bcl-2, which is an intracellular membrane protein shown to block or delay apoptosis. Overexpression of bcl-2 has been shown to be related to hyperplasia, autoimmunity and resistance to apoptosis, including that induced by chemotherapy (Fang et al., J. Immunol. 1994, 153, 4388-4398). A family of bcl-2-related genes has been described. All bcl-2 family members share two highly conserved domains, BH1 and BH2. bcl-2 family members include, but are not limited to, A1, mcl-1, bcl-w, bax, bad, bak and bcl-x. A1, mcl-1, bcl-w and bcl-xl (long form of bcl-x) are presently known to confer protection against apoptosis and are referred to herein as "anti-apoptotic bcl-2-related proteins." Of these, A1 and mcl-1 are known as "novel anti-apoptotic bcl-2-related proteins." In contrast, bax, bad, bak and bcl-xs (short form of bcl-x) are presently known to promote cell death by inhibiting this protective effect. The present invention relates to the novel anti-apoptotic human bcl-2-related proteins, particularly human A1 and mcl-1, and inhibition of the expression of these proteins using antisense technology.
The gene encoding A1 (also known as bcl-2-related gene expressed in fetal liver, or bfl-1, and Glasgow Rearranged Sequence, or GRS) was identified as an early response gene in murine hematopoietic cells treated with granulocyte-macrophage colony-stimulating factor. The human homolog was subsequently found to have extensive homology to bcl-2, especially within the BH1 and BH2 domains. A correlation was noted between the expression level of A1 and the development of stomach cancer in clinical samples. Choi et al., 1995, Oncogene 11, 1693-1698; WO 96/30513. The coding sequence of human A1 was cloned (Karsan et al., 1996, Blood 87, 3089-3096; Genbank accession no. U29680) and found to be expressed by hematopoietic cells but also a variety of nonhematopoietic tissues. A1 is rapidly inducible by phorbol esters and inflammatory cytokines, and possibly by vascular endothelial growth factor. A1 is believed to play a role in the regulation of physiological cell death during embryonic development. Carrio et al., 1996, Am. J. Path., 149, 2133-2142. There is slight variation among A1 sequences, with the human A1 sequence originally defined as "GRS" differing by two amino acids from the A1 sequence originally defined as "bfl-1" The GRS sequence was originally isolated by NIH3T3 focus formation assay using DNA obtained from a patient with chronic myeloid leukemia. Kenny et al, Oncogene 14, 1997 997-1001. These researchers found a high level of A1 expression in the cancer cell lines U-937 (histiocytic lymphoma), HL-60 (promyelocytic leukemia) and Raji (Burkitt lymphoma) cells. Expression of A1 was also found in THP-1 (acute myeloid leukemia), BJAB (Burkitt lymphoma), activated Jurkat (acute T-cell leukemia) cells and K-562 erythroleukemia (CML blast crisis) cells.
mcl-1 was originally identified from the differentiating human myeloid leukemia cell line ML-1. Its expression was found to increase early in the induction or "programming" of differentiation of ML-1 cells before the appearance of differentiation markers. The coding region of mcl-1 was sequenced and found to have a pronounced region of sequence homology to bcl-2 in the carboxyl-terminal region. Kozopas et al., Proc. Natl. Acad. Sci. USA., 1993, 90, 3516-3520; Genbank accession no. L08246. Unlike bcl-2, mcl-1 contains a strong PEST sequence (enriched in proline, glutamic acid, serine and threonine) which is present in a variety of proteins that undergo rapid turnover.
Overexpression of exogenously introduced mcl-1 has been shown to cause a prolongation of viability under conditions that normally cause apoptotic cell death, such as exposure to cytotoxic agents (the chemotherapeutic agent etoposide, calcium ionophore or UV irradiation) or the withdrawal of required growth factors. Zhou et al., Blood 89, 1997, 630-643.
Diseases and conditions in which apoptosis has been implicated fall into two categories, those in which there is increased cell survival (i.e., apoptosis is reduced) and those in which there is excess cell death (i.e., apoptosis is increased). Diseases in which there is an excessive accumulation of cells due to increased cell survival include cancer, autoimmune disorders and viral infections. Until recently, it was thought that cytotoxic drugs killed target cells directly by interfering with some life-maintaining function. However, of late, it has been shown that exposure to several cytotoxic drugs with disparate mechanisms of action induces apoptosis in both malignant and normal cells. Manipulation of levels of trophic factors (e.g., by anti-estrogen compounds or those which reduce levels of various growth hormones) has been one clinical approach to promote apoptosis, since deprivation of trophic factors can induce apoptosis. Apoptosis is also essential for the removal of potentially autoreactive lymphocytes during development and the removal of excess cells after the completion of an immune or inflammatory response. Recent work has clearly demonstrated that improper apoptosis may underlie the pathogenesis of autoimmune diseases by allowing abnormal autoreactive lymphocytes to survive. For these and other conditions in which insufficient apoptosis is believed to be involved, promotion of apoptosis is desired. Inhibition of novel anti-apoptotic bcl-2-related proteins according to the present invention is believed to result in promotion of apoptosis.
In the second category, AIDS and neurodegenerative disorders like Alzheimer's or Parkinson's disease represent disorders for which an excess of cell death due to promotion of apoptosis (or unwanted apoptosis) has been implicated. Amyotrophic lateral sclerosis, retinitis pigmentosa, and epilepsy are other neurologic disorders in which apoptosis has been implicated. Apoptosis has been reported to occur in conditions characterized by ischemia, e.g. myocardial infarction and stroke. Apoptosis has also been implicated in a number of liver disorders including obstructive jaundice and hepatic damage due to toxins and drugs. Apoptosis has also been identified as a key phenomenon in some diseases of the kidney, i.e. polycystic kidney, as well as in disorders of the pancreas including diabetes (Thatte, et al., Drugs, 1997, 54, 511-532). For these and other diseases and conditions in which unwanted apoptosis is believed to be involved, inhibitors of apoptosis are desired.
Antisense oligonucleotides have been used to elucidate the role of several members of the bcl-2 family. Extensive studies using antisense oligonucleotides targeted to bcl-2 have been performed, and an antisense compound (G3139, Genta Incorporated) targeted to human bcl-2 has entered clinical trials for lymphoma and prostate cancer.
Amarante-Mendes et al., Oncogene, 1998, 16, 1383-1390, disclose antisense oligonucleotides targeted to bcr and bcl-x. The latter downregulated the expression of bcl-xl and increased the susceptibility of HL-60 Bcr-Abl cells to staurosporine.
U.S. Pat. No. 5,583,034 (Green et al.) discloses antisense oligonucleotides which hybridize to the nucleic acid sequence of an anti-apoptotic gene, preferably to the translation start site of bcr-abl.
Wang et al. used a phosphorothioate oligonucleotide targeted to the bcl-x translation start site to block CD40L-mediated apoptotic rescue in murine WEHI-231 lymphoma cells (J. Immunol., 1995, 155, 3722-3725).
Fujio et al. have used an antisense oligodeoxynucleotide targeted to murine and rat bcl-x mRNA to reduce bcl-xl protein expression (J. Clin. Invest., 1997, 99, 2898-2905). The compound tested was the same as that of Wang et al. Oligonucleotide treatment inhibited the cytoprotective effect of leukemia inhibitory factor in mouse or rat cardiac myocytes.
Pollman et al. used antisense oligodeoxynucleotides with phosphorothioate backbones to downregulate bcl-xl expression in blood vessel intimal cells (Nature Med., 1998, 4, 222-227). This resulted in induction of apoptosis and regression of vascular lesions. Antisense sequences were targeted to the translation initiation codon of mouse/human bcl-x (conserved sequence) and were used in rabbits. Gibbons et al., U.S. Pat. No. 5,776,905, disclose methods for targeted deletion of intimal lesion cells in the vasculature of a mammal with vascular disease, preferably with antisense molecules specific for anti-apoptotic genes, more preferably bcl-x and most preferably bcl-xl.
Thompson et al., U.S. Pat. No. 5,646,008 and WO 95/00642 describe an isolated and purified polynucleotide that encodes a polypeptide other than bcl-2 that promotes or inhibits programmed vertebrate cell death. Preferably the polypeptide is bcl-xl, bcl-xs or bcl-x.sub.1. Polypeptides, polynucleotides identical or complementary to a portion of the isolated and purified polynucleotide, expression vectors, host cells, antibodies and therapeutic and diagnostic methods of use are also provided.
Yang et al., WO 98/05777 disclose bcl-x.gamma. (gamma), a novel isoform of the bcl-x family which includes an ankyrin domain. Polypepticde and nucleic acid sequences for this isoform are disclosed, as well as, inter alia, methods for modulating bcl-x.gamma. activity, including antisense methods.
Chao et al., Molec. Cell. Biol., 1998, 18, 4883-4898, used an antisense construct containing the entire human mcl-1 cDNA in antisense orientation to show that down-regulation of endogenous mcl-1 in TF-1 cells can induce apoptosis of these cells.
Cory et al. (WO 97/35971) disclose methods for modulating expression of bcl-w in a mammal by contacting the bcl-w gene with an effective amount of a modulator of bcl-w expression. Both enhanced and decreased bcl-w expression, including use of antisense sequences to bcl-w, are disclosed.
WO 96/30513 (Shin et al.) discloses the sequence of a Bcl-2 related gene, Bfl-1, and use of a Bcl-2 related gene for diagnosing cancer.
WO 94/29330 discloses, inter alia, the mcl-1 polypeptide sequence and polynucleotide sequence, host cells and vectors containing the latter, antibodies which bind to the mcl-1 polypeptide, and diagnostic and therapeutic methods using these compounds. Methods for treating a subject with a mcl-1 associated cell proliferative disorder are generally disclosed, including antisense oligonucleotide and ribozyme approaches. No specific sequence or targeting information is provided.